ABSTRACT Advances in molecular biology and genetic engineering have led to the design and use of modified T cells recognize tumors to achieve significant tumor control upon adoptive cell transfer (ACT) to patients. These T cells are either transduced with tumor antigen reactive T cell receptors (TCR), or chimeric antigen receptors (CARs). Recently, a surge in studies with neo-antigen reactive T cells or T cells recognizing novel mutated antigens has also shown promise. While the implementation of these studies requires significant technical resources, the appearance of antigen loss variants also leads to less effective tumor control when using effector T cells reactive to single tumor antigen or target molecule. Thus, there is a resurgence in using TILs, which have endogenous T cells reactive to multiple tumor epitopes, for ACT. While most studies have used the conventional approach to expand TILs using high dose IL2, some recent studies using IL15 or IL21 showed improved tumor control. Preclinical studies have also shown that different subsets of both helper CD4+ T helper (Th) cells and CD8+ T cytotoxic (Tc) cells hold promise for clinical use in ACT protocols. Importantly, T helper cell subsets with the ability to secrete IL-17 (Th17) have been shown to possess stem cell like phenotype that attributes to their long-term persistence and leads to improved tumor control tumors as compared to the Th1 subsets (that secrete IFN?, IL2, TNF?). However, contrary to these observations there are reports that Tc1 cells exhibit improved tumor control as compared to Tc17 cells. These differences in T cell subsets response to control tumors, is compounded by the fact that in the suppressive tumor microenvironment a large fraction of these Th or Tc subsets acquire FoxP3+ regulatory phenotype, become dysfunctional or undergo cell death leading to tumor reversion. Thus, ex vivo programming conditions that can render a stable phenotype with reduced `plasticity' and not only controls primary tumors, but also results in formation of anti-tumor memory will be of immense importance in ACT. We have recently established that programming conditions that bring together `anti-tumor effector function' of Th1 cells and `stemness' of Th17 cells lead to a superior hybrid Th1/17 (and Tc/17) cells exhibiting long-term tumor control. Thus, we hypothesize that ex vivo expansion and programming of TILs to hybrid T1/17 (Th1/17 and Tc1/17) phenotype will lead to robust anti-tumor control even with fewer adoptively transferred cells. Following specific aims are proposed to establish and develop our approach for commercialization: Specific Aim 1: To determine if human melanoma tumor derived TILs could be ex vivo programmed to potent anti-tumor hybrid T1/17 phenotype. Specific Aim 2: To establish if hybrid TILs are superior to conventional TILs in controlling melanoma tumor growth in vivo. We believe that this proposal will help adopt the novel ex vivo programming conditions for generating robust anti-tumor TILs that could be used future in adoptive T-cell immunotherapy clinical trials.